Computational modeling of epithelial–mesenchymal transformations

An epithelial–mesenchymal transformation (EMT) involves alterations in cell–cell and cell–matrix adhesion, the detachment of epithelial cells from their neighbors, the degradation of the basal lamina and acquisition of mesenchymal phenotype. Here we present Monte Carlo simulations for a specific EMT in early heart development: the formation of cardiac cushions. Cell rearrangements are described in accordance with Steinberg's differential adhesion hypothesis, which states that cells possess a type-dependent adhesion apparatus and are sufficiently motile to give rise to the tissue conformation with the largest number of strong bonds. We also implement epithelial and mesenchymal cell proliferation, cell type change and extracellular matrix production by mesenchymal cells. Our results show that an EMT is promoted more efficiently by an increase in cell–substrate adhesion than by a decrease in cell–cell adhesion. In addition to cushion tissue formation, the model also accounts for the phenomena of matrix invasion and mesenchymal condensation. We conclude that in order to maintain epithelial integrity during EMT the number of epithelial cells must increase at a controlled rate. Our model predictions are in qualitative agreement with available experimental data.     

Extracellular matrix proteins regulate epithelial–mesenchymal transition in mammary epithelial cells

Mouse mammary epithelial cells undergo transdifferentiation via epithelial–mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland.     

Cellular contractility changes are sufficient to drive epithelial scattering

Epithelial scattering occurs when cells disassemble cell–cell junctions, allowing individual epithelial cells to act in a solitary manner. Epithelial scattering occurs frequently in development, where it accompanies epithelial–mesenchymal transitions and is required for individual cells to migrate and invade. While migration and invasion have received extensive research focus, how cell–cell junctions are detached remains poorly understood. An open debate has been whether disruption of cell–cell interactions occurs by remodeling of cell–cell adhesions, increased traction forces through cell substrate adhesions, or some combination of both processes. Here we seek to examine how changes in adhesion and contractility are coupled to drive detachment of individual epithelial cells during hepatocyte growth factor (HGF)/scatter factor-induced EMT. We find that HGF signaling does not alter the strength of cell–cell adhesion between cells in suspension, suggesting that changes in cell–cell adhesion strength might not accompany epithelial scattering. Instead, cell–substrate adhesion seems to play a bigger role, as cell–substrate adhesions are stronger in cells treated with HGF and since rapid scattering in cells treated with HGF and TGFβ is associated with a dramatic increase in focal adhesions. Increases in the pliability of the substratum, reducing cells ability to generate traction on the substrate, alter cells? ability to scatter. Further consistent with changes in substrate adhesion being required for cell–cell detachment during EMT, scattering is impaired in cells expressing both active and inactive RhoA mutants, though in different ways. In addition to its roles in driving assembly of both stress fibers and focal adhesions, RhoA also generates myosin-based contractility in cells. We therefore sought to examine how RhoA-dependent contractility contributes to cell–cell detachment. Inhibition of Rho kinase or myosin II induces the same effect on cells, namely an inhibition of cell scattering following HGF treatment. Interestingly, restoration of myosin-based contractility in blebbistatin-treated cells results in cell scattering, including global actin rearrangements. Scattering is reminiscent of HGF-induced epithelial scattering without a concomitant increase in cell migration or decrease in adhesion strength. This scattering is dependent on RhoA, as blebbistatin-induced scattering is reduced in cells expressing dominant-negative RhoA mutants. This suggests that induction of myosin-based cellular contractility may be sufficient for cell–cell detachment during epithelial scattering.     

Regulation of Cell–Cell Adhesion by Rac and Rho Small G Proteins in MDCK Cells

The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton. We show here that the Rac and Rho subfamilies furthermore regulate cell–cell adhesion. We prepared MDCK cell lines stably expressing each of dominant active mutants of RhoA (sMDCK-RhoDA), Rac1 (sMDCK-RacDA), and Cdc42 (sMDCK-Cdc42DA) and dominant negative mutants of Rac1 (sMDCK-RacDN) and Cdc42 (sMDCK-Cdc42DN) and analyzed cell adhesion in these cell lines. The actin filaments at the cell–cell adhesion sites markedly increased in sMDCK-RacDA cells, whereas they apparently decreased in sMDCK-RacDN cells, compared with those in wild-type MDCK cells. Both E-cadherin and β-catenin, adherens junctional proteins, at the cell–cell adhesion sites also increased in sMDCK-RacDA cells, whereas both of them decreased in sMDCK-RacDN cells. The detergent solubility assay indicated that the amount of detergent-insoluble E-cadherin increased in sMDCK-RacDA cells, whereas it slightly decreased in sMDCK-RacDN cells, compared with that in wild-type MDCK cells. In sMDCK-RhoDA, -Cdc42DA, and -Cdc42DN cells, neither of these proteins at the cell–cell adhesion sites was apparently affected. ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines. Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes. These results suggest that the Rac subfamily regulates the formation of the cadherin-based cell– cell adhesion. Microinjection of C3 into wild-type MDCK cells inhibited the formation of both the cadherin-based cell–cell adhesion and the tight junction, but microinjection of C3 into sMDCK-RacDA cells showed little effect on the localization of the actin filaments and E-cadherin at the cell–cell adhesion sites. These results suggest that the Rho subfamily is necessary for the formation of both the cadherin-based cell– cell adhesion and the tight junction, but not essential for the Rac subfamily-regulated, cadherin-based cell– cell adhesion.     

Celastrol inhibits TGF-β1-induced epithelial–mesenchymal transition by inhibiting Snail and regulating E-cadherin expression

The epithelial–mesenchymal transition (EMT) is a pivotal event in the invasive and metastatic potentials of cancer progression. Celastrol inhibits the proliferation of a variety of tumor cells including leukemia, glioma, prostate, and breast cancer; however, the possible role of celastrol in the EMT is unclear. We investigated the effect of celastrol on the EMT. Transforming growth factor-beta 1 (TGF-β1) induced EMT-like morphologic changes and upregulation of Snail expression. The downregulation of E-cadherin expression and upregulation of Snail in Madin–Darby Canine Kidney (MDCK) and A549 cell lines show that TGF-β1-mediated the EMT in epithelial cells; however, celastrol markedly inhibited TGF-β1-induced morphologic changes, Snail upregulation, and E-cadherin expression. Migration and invasion assays revealed that celastrol completely inhibited TGF-β1-mediated cellular migration in both cell lines. These findings indicate that celastrol downregulates Snail expression, thereby inhibiting TGF-β1-induced EMT in MDCK and A549 cells. Thus, our findings provide new evidence that celastrol suppresses lung cancer invasion and migration by inhibiting TGF-β1-induced EMT.     

Numb

Epithelial to mesenchymal transition (EMT) is a critical event in embryogenesis and plays a fundamental role in cancer progression and metastasis1. Numb has been shown to play an important role in the proper functions of Par protein complex and in cell-cell junctions, both of which are associated with EMT. However, the role of Numb in EMT has not been fully elucidated. Recently, we showed that Numb is capable of binding to both Par3 and E-cadherin. Intriguingly, the interaction of Numb with E-cadherin or the Par protein complex is dynamically regulated by tyrosine phosphorylation induced by HGF or Src. Knockdown of Numb by shRNA in MDCK cells led to an lateral to apical translocation of E-cadherin and β-catenin, active F-actin polymerization, mis-localization of Par3 and aPKC, a decrease in cell-cell adhesion, and an increase in cell migration and proliferation. These data suggest a diverse role for Numb in regulating cell-cell adhesion, polarity and migration during EMT.     

XLMR protein related to neurite extension (Xpn/KIAA2022) regulates cell–cell and cell–matrix adhesion and migration

X-linked mental retardation (XLMR) is a common cause of moderate to severe intellectual disability in males. XLMR protein related to neurite extension (Xpn, also known as KIAA2022) has been implicated as a gene responsible for XLMR in humans. Although Xpn is highly expressed in the developing brain and is involved in neurite outgrowth in PC12 cells and neurons, little is known about the functional role of Xpn. Here, we show that Xpn regulates cell–cell and cell–matrix adhesion and migration in PC12 cells. Xpn knockdown enhanced cell–cell and cell–matrix adhesion mediated by N-cadherin and β1-integrin, respectively. N-Cadherin and β1-integrin expression at the mRNA and protein levels was significantly increased in Xpn knockdown PC12 cells. Furthermore, overexpressed Xpn protein was strongly expressed in the nuclei of PC12 and 293T cells. Finally, depletion of Xpn perturbed cellular migration by enhancing N-cadherin and β1-integrin expression in a PC12 cell wound healing assay. We conclude that Xpn regulates cell–cell and cell–matrix adhesion and cellular migration by regulating the expression of adhesion molecules.     

Competitive enhancement of HGF-induced epithelial scattering by accessory growth factors

HGF signaling induces epithelial cells to disassemble cadherin-based adhesion and increase cell motility and invasion, a process termed epithelial–mesenchymal transition (EMT). EMT plays a major role in cancer metastasis, allowing individual cells to detach from the primary tumor, invade local tissue, and colonize distant tissues with new tumors. While invasion of vascular and lymphatic networks is the predominant route of metastasis, nerves also can act as networks for dissemination of cancer cell to distant sites in a process termed perineual invasion (PNI). Signaling between nerves and invasive cancer cells remains poorly understood, as does cellular decision making that selects the specific route of invasion. Here we examine how HGF signaling contributes to PNI using reductionist culture model systems. We find that TGFβ, produced by PC12 cells, enhances scattering in response to HGF stimulation, increasing both cell–cell junction disassembly and cell migration. Further, gradients of TGFβ induce migratory mesenchymal cells to undergo chemotaxis towards the source of TGFβ. Interestingly, VEGF suppresses TGFβ-induced enhancement of scattering. These results have broad implications for how combinatorial growth factor signaling contributes to cancer metastasis, suggesting that VEGF and TGFβ might modulate HGF signaling to influence route selection during cancer progression.     

Matrix-dependent Tiam1/Rac Signaling in Epithelial Cells Promotes Either Cell–Cell Adhesion or Cell Migration and Is Regulated by Phosphatidylinositol 3-Kinase

We previously demonstrated that both Tiam1, an activator of Rac, and constitutively active V12Rac promote E-cadherin–mediated cell–cell adhesion in epithelial Madin Darby canine kidney (MDCK) cells. Moreover, Tiam1 and V12Rac inhibit invasion of Ras-transformed, fibroblastoid MDCK-f3 cells by restoring E-cadherin–mediated cell–cell adhesion. Here we show that the Tiam1/Rac-induced cellular response is dependent on the cell substrate. On fibronectin and laminin 1, Tiam1/Rac signaling inhibits migration of MDCK-f3 cells by restoring E-cadherin–mediated cell– cell adhesion. On different collagens, however, expression of Tiam1 and V12Rac promotes motile behavior, under conditions that prevent formation of E-cadherin adhesions. In nonmotile cells, Tiam1 is present in adherens junctions, whereas Tiam1 localizes to lamellae of migrating cells. The level of Rac activation by Tiam1, as determined by binding to a glutathione-S-transferase– PAK protein, is similar on fibronectin or collagen I, suggesting that rather the localization of the Tiam1/Rac signaling complex determines the substrate-dependent cellular responses. Rac activation by Tiam1 requires PI3-kinase activity. Moreover, Tiam1- but not V12Rac-induced migration as well as E-cadherin–mediated cell– cell adhesion are dependent on PI3-kinase, indicating that PI3-kinase acts upstream of Tiam1 and Rac.     

Hypoxia Activated EGFR Signaling Induces Epithelial to Mesenchymal Transition (EMT)

Metastasis is a multi-step process which requires the conversion of polarized epithelial cells to mesenchymal cells, Epithelial–Mesenchymal Transition (EMT). EMT is essential during embryonic morphogenesis and has been implicated in the progression of primary tumors towards metastasis. Hypoxia is known to induce EMT; however the molecular mechanism is still poorly understood. Using the A431 epithelial cancer cell line, we show that cells grown under hypoxic conditions migrated faster than cells grown under normal oxygen environment. Cells grown under hypoxia showed reduced adhesion to the extracellular matrix (ECM) probably due to reduced number of Vinculin patches. Growth under hypoxic conditions also led to down regulation of E-cadherin and up regulation of vimentin expression. The increased motility of cells grown under hypoxia could be due to redistribution of Rac1 to the plasma membrane as opposed to increased expression of Rac1. EGF (Epidermal Growth Factor) is a known inducer of EMT and growth of A431 cells in the absence of oxygen led to increased expression of EGFR (EGF Receptor). Treatment of A431 cells with EGF led to reduced cell adhesion to ECM, increased cell motility and other EMT characteristics. Furthermore, this transition was blocked by the monoclonal antibody Cetuximab. Cetuximab also blocked the hypoxia-induced EMT suggesting that cell growth under hypoxic conditions led to activation of EGFR signaling and induction of EMT phenotype.     

Inhibitory effects of ceramide kinase on Rac1 activation,lamellipodium formation,cell migration,and metastasis of A549 lung cancer cells

Ceramide kinase (CerK) phosphorylates ceramide to ceramide-1-phosphate (C1P), a bioactive sphingolipid. Since the mechanisms responsible for regulating the proliferation and migration/metastasis of cancer cells by the CerK/C1P pathway remain unclear, we conducted the present study. The knockdown of CerK in A549 lung and MCF-7 breast cancer cells (shCerK cells) increased the formation of lamellipodia, which are membrane protrusions coupled with cell migration. Mouse embryonic fibroblasts prepared from CerK-null mice also showed an enhanced formation of lamellipodia. The overexpression of CerK inhibited lamellipodium formation in A549 cells. The knockdown of CerK increased the number of cells having lamellipodia with Rac1 and the levels of active Rac1-GTP form, whereas the overexpression of CerK decreased them. CerK was located in lamellipodia after the epidermal growth factor treatment, indicating that CerK functioned there to inhibit Rac1. The migration of A549 cells was negatively regulated by CerK. An intravenous injection of A549-shCerK cells into nude mice resulted in markedly stronger metastatic responses in the lungs than an injection of control cells. The in vitro growth of A549 cells and in vivo expansion after the injection into mouse flanks were not affected by the CerK knockdown. These results suggest that the activation of CerK/C1P pathway has inhibitory roles on lamellipodium formation, migration, and metastasis of A549 lung cancer cells.     

Establishment of Hertwig's epithelial root sheath cell line from cells involved in epithelial-mesenchymal transition

The epithelial–mesenchymal transition (EMT) is an important event in the developmental process of various organs. In periodontal development during root formation of a tooth, this EMT has been a subject of controversy. Hertwig’s epithelial root sheath (HERS), consisting of two epithelial layers, plays a role of inducing odontogenesis during root development and thereafter becomes fragmented. Some researchers have maintained that in the process of this fragmentation, some HERS cells change from epithelial to mesenchymal cells. Here, we established a HERS cell line (HERS01a) and examined its gene and protein expression. Immunohistochemical staining and real-time PCR analysis showed that HERS01a cells expressed vimentin and N-cadherin as mesenchymal markers as well as cytokeratin14, E-cadherin, and p63 as epithelial stem cell markers. In the presence of TGF-β, HERS01a cells also expressed many more mesenchymal markers, as well as snail1 and 2 as EMT markers. Taken together, our data show that HERS01a displayed unique features associated with EMT in the root formation process, and will thus be useful for analyzing the biological characteristics of HERS and the molecular mechanism underlying the EMT.     

Epithelial–mesenchymal transition in cancer metastasis: Mechanisms,markers and strategies to overcome drug resistance in the clinic

Epithelial–mesenchymal transition (EMT) is a key step during embryogenesis. Accumulating evidence suggests a critical role in cancer progression, through which tissue epithelial cancers invade and metastasise. Cell characteristics are highly affected during EMT, resulting in altered cell–cell and cell–matrix interactions, cell motility and invasiveness. Nevertheless, the demonstration of this process in human cancer has been proven difficult and controversial. Besides the fact that the acquisition of mesenchymal characteristics is not a prerequisite for cell migration/invasion, it is a transient event that concerns only few cells in a tumour mass. The induction of EMT depends on the tumour type and its genetic alterations as well as on its interaction with the extracellular matrix. In parallel, trials for EMT identification in clinical samples lack of a widely accepted methodology, nomenclature and reliable markers. This review summarizes the main EMT characteristics and proposes methodologies for better analysis in vitro. It also highlights recent studies identifying cells with EMT characteristics in human cancer and proposes certain markers to identify them in tumour samples. Finally, it cites the recent literature concerning the mechanisms of drug resistance related to EMT in the context of anti-tumour therapies and proposes related new targets for therapy.     

N1-guanyl-1,7-diaminoheptane (GC7) enhances the therapeutic efficacy of doxorubicin by inhibiting activation of eukaryotic translation initiation factor 5A2 (eIF5A2) and preventing the epithelial–mesenchymal transition in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) cells undergo the epithelial–mesenchymal transition (EMT) during chemotherapy, which reduces the efficacy of doxorubicin-based chemotherapy. We investigated N1-guanyl-1,7-diaminoheptane (GC7) which inhibits eukaryotic translation initiation factor 5A2 (eIF5A2) activation; eIF5A2 is associated with chemoresistance. GC7 enhanced doxorubicin cytotoxicity in epithelial HCC cells (Huh7, Hep3B and HepG2) but had little effect in mesenchymal HCC cells (SNU387, SNU449). GC7 suppressed the doxorubicin-induced EMT in epithelial HCC cells; knockdown of eIF5A2 inhibited the doxorubicin-induced EMT and enhanced doxorubicin cytotoxicity. GC7 combination therapy may enhance the therapeutic efficacy of doxorubicin in HCC by inhibiting eIF5A2 activation and preventing the EMT.     

Cigarette smoke-induced alveolar epithelial–mesenchymal transition is mediated by Rac1 activation

Background

Epithelial–mesenchymal transition (EMT) is the major pathophysiological process in lung fibrosis observed in chronic obstructive pulmonary disease (COPD) and lung cancer. Smoking is a risk factor for developing EMT, yet the mechanism remains largely unknown. In this study, we investigated the role of Rac1 in cigarette smoke (CS) induced EMT.

Methods

EMT was induced in mice and pulmonary epithelial cells by exposure of CS and cigarette smoke extract (CSE) respectively.

Results

Treatment of pulmonary epithelial cells with CSE elevated Rac1 expression associated with increased TGF-β1 release. Blocking TGF-β pathway restrained CSE-induced changes in EMT-related markers. Pharmacological inhibition or knockdown of Rac1 decreased the CSE exposure induced TGF-β1 release and ameliorated CSE-induced EMT. In CS-exposed mice, pharmacological inhibition of Rac1 reduced TGF-β1 release and prevented aberrations in expression of EMT markers, suggesting that Rac1 is a critical signaling molecule for induction of CS-stimulated EMT. Furthermore, Rac1 inhibition or knockdown abrogated CSE-induced Smad2 and Akt (PKB, protein kinase B) activation in pulmonary epithelial cells. Inhibition of Smad2, PI3K (phosphatidylinositol 3-kinase) or Akt suppressed CSE-induced changes in epithelial and mesenchymal marker expression.

Conclusions and general significance

Altogether, these data suggest that CS initiates EMT through Rac1/Smad2 and Rac1/PI3K/Akt signaling pathway. Our data provide new insights into the fundamental basis of EMT and suggest a possible new course of therapy for COPD and lung cancer.     

miR-181a–Twist1 pathway in the chemoresistance of tongue squamous cell carcinoma

Although many researches have been undertaken to disclose the mechanisms of chemoresistance, the mechanisms remain unclear. The aim of this study is to elucidate the role of miR-181a–Twist1 pathway in the chemoresistance of tongue squamous cell carcinoma (TSCC). We found that cisplatin-induced chemoresistance in TSCC cell lines underwent EMT (epithelial–mesenchymal transition) and was accompanied by enhancing metastatic potential (migration and invasion in vitro), miR-181a downregulation and Twist1 upregulation. Functional analyses indicated that miR-181a reversed chemoresistance, inhibited EMT and metastatic potential in TSCC cells. Twist1 was confirmed as a direct miR-181a target gene by luciferase reporter gene assays. Twist1 knockdown by siRNA led to a reversal of the chemoresistance, inhibited EMT and metastatic potential in TSCC cells. Our study demonstrates that miR-181a–Twist1 pathway may play an important role in the development of cisplatin-chemoresistance, with EMT and an increase the metastatic potential of TSCC cells.     

The Small GTPases Rho and Rac Are Required for the Establishment of Cadherin-dependent Cell–Cell Contacts

Cadherins are calcium-dependent cell–cell adhesion molecules that require the interaction of the cytoplasmic tail with the actin cytoskeleton for adhesive activity. Because of the functional relationship between cadherin receptors and actin filament organization, we investigated whether members of the Rho family of small GTPases are necessary for cadherin adhesion. In fibroblasts, the Rho family members Rho and Rac regulate actin polymerization to produce stress fibers and lamellipodia, respectively. In epithelial cells, we demonstrate that Rho and Rac are required for the establishment of cadherin-mediated cell–cell adhesion and the actin reorganization necessary to stabilize the receptors at sites of intercellular junctions. Blocking endogenous Rho or Rac selectively removed cadherin complexes from junctions induced for up to 3 h, while desmosomes were not perturbed. In addition, withdrawal of cadherins from intercellular junctions temporally precedes the removal of CD44 and integrins, other microfilament-associated receptors. Our data showed that the concerted action of Rho and Rac modulate the establishment of cadherin adhesion: a constitutively active form of Rac was not sufficient to stabilize cadherindependent cell–cell contacts when endogenous Rho was inhibited. Upon induction of calcium-dependent intercellular adhesion, there was a rapid accumulation of actin at sites of cell–cell contacts, which was prevented by blocking cadherin function, Rho or Rac activity. However, if cadherin complexes are clustered by specific antibodies attached to beads, actin recruitment to the receptors was perturbed by inhibiting Rac but not Rho. Our results provide new insights into the role of the small GTPases in the cadherin-dependent cell– cell contact formation and the remodelling of actin filaments in epithelial cells.